PROJECT SUMMARY Quantitative information on biomarkers is critical when certain clinical, public safety, and/or legislative decisions are to be made. Currently existing technologies are rather complex and, as a result, expensive. These characteristics limit availability of quantitative assessment in some situations where knowledge of biomarkers? amounts is of vital importance. Our team have recently developed a new platform for quantitative analysis of biomarkers: Quantitation without Calibration (QwiC). The platform addresses two major deficiencies in quantitative analysis: ? need for biomarker?s standard and calibration and ? uncertainties associated with signal magnitude variations. QwiC relies on position of target?s response profile to get quantitative information on a target. The platform rests upon a major innovation: a deliberate incorporation of negative cooperativity into probe-target binding interactions. The incorporation of the mechanism allows assessing target quantity from its response profile instead of relying on signal magnitude and calibration. In order to further advance the methodology, we will accomplish two major goals: (i) Establish and characterize the target/probe recognition/binding model based on negative cooperativity as a platform for quantitative analysis of oligonucleotide targets and (ii) Establish and characterize the target-probe recognition/binding model based on negative cooperativity as a platform for quantitative analysis of antibodies. The development of the QwiC platform will transform an analytical measurement practice. Thus, simplification of getting quantitative data will result in better understanding of process in living systems and beyond. Besides, as an inexpensive, robust, and simple separation?less approach, QwiC will ultimately be a valuable asset for point-of-care, limited resources, and/or high-throughput environments. Furthermore, the project will enhance research environment in the Northern Illinois University by giving opportunity to generations of undergraduate students to get hands on experience with development of new methodologies and with a broad spectrum of modern bioanalytical techniques.